Method of reducing thrombosis and complications after arterial angioplasty using stent coated with magnesium-based compound

ABSTRACT

Treatment with magnesium produces a inhibition of acute stent thrombosis under high-shear flow conditions without any hemostatic or significant hemodynamic complications.

This application claims the benefit of priority under 35 U.S.C. §111 asa divisional of U.S. patent application Ser. No. 09/908,342, filed Jul.18, 2001, now issued as U.S. Pat. No. 6,692,772, on Feb. 17, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods of treating thrombosis, and moreparticularly to the use of magnesium to prevent conditions such asin-stent thrombosis.

2. Discussion of the Related Art

Coronary artery disease is one of the country's largest health concerns.According to the American Heart Association, this disease affects 13.5million Americans. Almost a million of these people have experiencedheart attacks. Still others have experienced angina, undergone coronaryartery bypass surgery and/or had heart transplants. Others in the later,or more severe, stages of coronary artery disease are in varying stagesof congestive heart failure.

Coronary artery disease, which has been linked with the increase incholesterol and saturated fat in our diets, is treatable. One mainsymptom of coronary artery disease is the deposit of these fattymaterials alongside a vessel wall such as an arterial wall. This resultsin the progressive narrowing of the lumen, and arteriosclerosis. Suchdeposits may be treated through a procedure called angioplasty. Duringangioplasty, the doctor inserts a catheter into an artery, typically agroin artery, and maneuvers the catheter up through the artery until thecatheter is positioned at the site of the narrowing or obstructioncaused by plaque. The plaque may then be flattened by inflating aballoon located around the tip of the catheter. As the balloon expands,it compresses the fatty deposits against the walls of the artery.

During this potentially lifesaving procedure, the doctor may insert astent into the vessel at the site of the blockage. This small, typicallymetallic device helps to hold the vessel open and improves blood flow.This serves to relieve the symptoms of Coronary Artery Disease.

Unfortunately, stents do not provide an absolute solution to thisproblem. Restenosis, a narrowing of the passageway which may initiatedby platelet adhesion and aggregation at the site of arterial injury, andthrombosis frequently occur at the site of the stent. “Thrombosis”describes the formation of a thrombus, or blood clot, inside a bloodvessel. Thrombosis may be caused by the continuous stresses from bloodflow over the stent. The longitudinal lumen of a stent usually, if notalways, has an irregular surface or regions that protrude into the lumenthat can produce a turbulent fluid flow. Alternatively, the thrombosismay be the result of a foreign body reaction to the stent. The formationof a blood clot within a blood vessel may cause tissue damage. Such aclot may be life threatening, particularly when it partially orcompletely blocks the flow of blood through a blood vessel. Ifthrombosis occurs as a result of the stent placement, a secondaryprocedure or a surgical bypass operation is required.

Products such as aspirin, dipyridamole and heparin are known in the artto dissolve such clots. While these products may eliminate the clot,they have the potential serious side effect of causing prolongedbleeding. Additionally, the effect of the administration of suchproducts may only be reversed by the formation or addition of newplatelets.

It is desired to be able to place a stent within a vessel throughpercutaneous transluminal delivery without the risk of in-stentthrombosis. Such stent placement without the risk of in-stent thrombosiswould greatly increase the quality of life for many patients.Accordingly, a method to prevent the formation of in-stent thrombosisthat is not detrimental to the patient is desired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a perfusion protocol that was run in an ex-vivoporcine shunt model according to an embodiment of the present;

FIG. 2( a) provides bar graphs showing the effect on thrombus weight indifferent treatment groups in an experiment according to one embodimentof the present invention;

FIG. 2( b) provides views of nitinol stents (side-on view) depictingthrombus burden under the various treatment conditions;

FIG. 3 provides a graphical depiction of the effects of magnesium onheart rate and mean arterial blood pressure as shown by an experimentthat was conducted according to one embodiment of the present invention;

FIG. 4 provides a chart detailing experimental data collected from anexperiment run in accordance with one embodiment of the presentinvention; and

FIG. 5 provides a chart detailing the effects of the heparin andmagnesium as used in an experiment according to one embodiment of thepresent invention on 5 μg/mL collagen-induced platelet aggregation,bleeding time and ACT.

DETAILED DESCRIPTION

The present invention is the discovery that the administration ofmagnesium produces a significant time-dependent inhibition, andpotential prevention, of acute platelet-dependent stent thrombosis underhigh-shear flow conditions. This antithrombotic effect, which correlatesdirectly with serum magnesium ion levels, is primarily due to themagnesium's effect on platelets, magnesium's effect on coagulation andfibrinolysis.

An additional feature of the present invention is that the inhibition ofacute platelet-dependent stent thrombosis may be achieved without anyhemostatic or significant hemodynamic complications. Importantly, asignificant inhibition of platelet-thrombus formation was demonstratedwith magnesium treatment without any effect on platelet aggregation orbleeding time. The potent antithrombotic effects of magnesium togetherwith its safety, ease of administration, and low cost make it apromising treatment during percutaneous coronary intervention (PCI).

The timing of the magnesium administration according to the presentinvention is important to the inhibition of stent thrombosis. Themaximum antithrombotic effect of Mg is evident when platelets aretreated before exposure to thrombogenic stimuli. Such thrombogenicstimuli include, but are not limited to, catheters, endovasculardevices, stent-bearing catheters, angioplasty, stents, laser catheters,atherectomy, radiation, extraction devices (for example: transluminalextraction atherectomy), angiojets, and local drug-delivery catheters.

This time-dependent effect may be seen in FIG. 4, which provides datafrom an experiment conducted in accordance with one embodiment of thepresent invention. As shown in FIG. 4, the antithrombotic effects ofmagnesium were significantly more pronounced when Mg was given 40minutes, as opposed to 20 minutes, before initiation of stent perfusion.Given sufficient exposure to magnesium with appropriate timing, theadministration of magnesium may be used to assist in the prevention ofstent thrombosis.

The effects of the present invention are best seen when the magnesium isadministered between about 30 and 60 minutes prior to thromboticstimuli. However, it should be understood that the magnesium may beadministered within a reasonable time outside of this range. Forexample, the magnesium may be administered between 15 and 90 minutesprior to thrombotic stimuli. The magnesium may be administered, at leastin part, by coating a stent or other endoprosthesis with amagnesium-containing compound prior to insertion into a body passageway.Administration of the magnesium should be continued for approximatelyeight to twelve hours after the procedure.

In one embodiment of the present invention, magnesium is administeredintravenously. Intravenous administration allows for greater control ofthe level of magnesium in the blood. As should be understood by oneskilled in the art, however, the administration of magnesium is notlimited to intravenous administration. Other methods of administrationinclude, but are not limited to, oral ingestion and the administrationof an injection. Oral administration of magnesium may be particularlybeneficial to treat (or prevent) stent thrombosis in patients once theyare discharged. The magnesium may alternatively be supplied to a patientthrough a catheter or other instrument which is inserted percutaneously,or through a tube attached to such instrument. Such an instrument mayalternatively be coated with a magnesium-containing compound. In afurther embodiment of the present invention, a stent may be coated witha magnesium-containing compound.

The use of magnesium disclosed herein has been thus far limited to adiscussion of the inhibition of stent thrombosis. However, the presentinvention is not so limited. Magnesium may be administered as taught bythe present invention to prevent thrombosis during angioplasty andbypass and other surgeries.

The magnesium may be administered in the form of magnesium sulfate(MgSO₄), or any other form of magnesium known by those skilled in theart to be non-toxic to the subject. Such non-toxic forms of magnesiuminclude, but are not limited to, magnesium phosphate (MgPO₄), magnesiumchloride (MgCl) and magnesium oxide (MgO).

Magnesium has an anti-adhesive effect on platelets that is achievedprimarily by reducing calcium mobilization in platelets. It may alsosuppress fibrinogen interaction with platelets via competitiveinhibition of calcium at the calcium-binding sites of the glycoproteinIIb-IIIa complex. See Gawaz M, Ott I, Reininger A J, Neumann F J.Effects of magnesium on platelet aggregation and adhesion: magnesiummodulates surface expression of glycoproteins on platelets in vitro andex vivo. Thromb Haemost, 1994; 72:912-918. The serum Mg levels achievedin the example discussed herein (3.7±0.3 for heparin+Mg-early and3.8±0.4 for heparin+Mg-late) were in the range where the anti-adhesiveeffects would be more evident compared to the anti-aggregatory effect.

The discrepant effect on platelet adhesion/thrombus formation andplatelet aggregation may relate to the fact that the anti-adhesiveeffect of Mg may occur at a lower concentration (<4 mEq/L) compared tothe anti-aggregatory effect (>5 mEq/L). See Ravn H B, Kristensen S D,Hjortdal V E, Thygesen K, Husted S E, Early Administration ofIntravenous Magnesium Inhibits Arterial Thrombus Formation, ArteriosclerThromb Vasc Biol, 1997; 17:3620-3625; and Gawaz M, Ott I, Reininger A J,Neumann F J, Effects of magnesium on platelet aggregation and adhesion:magnesium modulates surface expression of glycoproteins on platelets invitro and ex vivo, Thromb Haemost, 1994; 72:912-918. Since the thrombusweights returned towards pre-treatment values during control perfusionruns post-treatment utilizing the same aortic strip, it is unlikely thatthe varying thrombogenicity of the porcine aortic strip may havecontributed to treatment effect.

The role that magnesium may take in the treatment of cardiovasculardiseases has not been determined. For example, recent clinical trialshave presented conflicting evidence about the role of magnesium in acutemyocardial infarction with the ISIS-4 trial (International Study ofInfarct Survival) showing no benefit (ISIS-4: A randomized factorialtrial assessing early oral captopril, oral mononitrate, and intravenousmagnesium sulfate in 58,050 patients with suspected myocardialinfarction. Lancet, 1995;345:669-685.) and the LIMIT-2 (LeicesterIntravenous Magnesium Intervention Trial) study providing strongevidence for a survival advantage. See Woods K L, Fletcher S, Roffe C,Haider Y. Intravenous magnesium sulphate in suspected acute myocardialinfarction: results of the second Leicester Intravenous MagnesiumIntervention Trial (LIMIT-2). Lancet, 1992:339:1553-8.

Experimental Model

An experiment according to one embodiment of the present invention wasperformed using an ex-vivo model that primarily examines shear-mediated,platelet-dependent thrombus formation. This experiment allowed for theevaluation of the effects of magnesium, and the time-dependent naturethereof, on acute platelet-dependent stent thrombosis in an ex-vivoporcine arteriovenous shunt model of high-shear blood flow.

Such a model is useful to study interaction of blood elements withstents and thrombogenic surfaces under controlled and well-definedconditions. This ex-vivo system was chosen for its reproducibility andsimplicity. It a sensitive tool to assess pre-clinical efficacy ofantithrombotic therapeutic interventions.

Animal Surgery

All procedures of the animal surgery conducted in conjunction with theexperiments discussed herein were performed in accordance with oneembodiment of the present invention were approved by the InstitutionalAnimal Care and Use Committee and conformed to the American HeartAssociation guidelines for animal research. Experiments were performedin 10 swine weighing 25 to 30 kg. After overnight fasting, swine weresedated with phenobarbital (5 mg/kg), and anesthesia was maintained with1% isoflurane after endotracheal intubation. The right carotid arteryand jugular vein were isolated and cannulated with 8F sheaths toestablish an extracorporeal circuit as described previously. See Kaul S,Makkar R R, Nakamura M, Litvack F, Shah P K, Forrester J S, Hutsell T,Eigler N L, Inhibition of Acute Stent Thrombosis under High-Shear FlowConditions by a Nitric Oxide Donor. DMHD/NO: An Ex-Vivo PorcineArteriovenous Shunt Study, Circulation, 1996, 94:2228-34. Arterial bloodgases and pH were monitored periodically and maintained at normal levelsby adjustment of the ventilation rate and tidal volume. Invasivearterial pressure measurement, oxygen saturation, ECG, and rectaltemperature were monitored continuously. A thermostatically controlledblanket was used to maintain temperature at 37° C. Venous blood wascollected for baseline platelet aggregation, complete blood cell count,and activated clotting time (ACT) measurements.

All animals received heparin at a dose of 10 U/kg as a bolus before thestudy to prevent thrombotic occlusion of catheters and tubing. Eachswine received an average of 200 U heparin, an amount that producesnegligible effects on thrombus formation at high-shear conditions inthis model. See Kaul S, Makkar R R, Nakamura M, Litvack F, Shah P K,Forrester J S, Hutsell T, Eigler N L, Inhibition of Acute StentThrombosis under High-Shear Flow Conditions by a Nitric Oxide Donor.DMHD/NO: An Ex-Vivo Porcine Arteriovenous Shunt Study. Circulation,1996, 94:2228-34. At the conclusion of the experiment, blood wascollected for complete blood cell counts, the carotid artery and jugularvein were ligated, and the animals were allowed to recover fromanesthesia before being returned to the vivarium. Each animal wasstudied twice with a minimum interval of 2 weeks between eachexperiment.

Coronary Stents

The stents tested were 7-mm-long slotted-tube-geometry devices made fromthe nickel-titanium alloy NITINOL (Advanced Coronary Technology, MenloPark, Calif.) (n=156 stents in 10 swine). Each stent weighed 24±3 mg andhad a strut thickness of 0.006 in. They had a silicon carbidegrit-blasted surface finish, which creates a uniform roughened surfaceknown to be highly thrombogenic in this model. See Kaul S, Makkar R R,Nakamura M, Litvack F, Shah P K, Forrester J S, Hutsell T, Eigler N L,Inhibition of Acute Stent Thrombosis under High-Shear Flow Conditions bya Nitric Oxide Donor. DMHD/NO: An Ex-Vivo Porcine Arteriovenous ShuntStudy. Circulation, 1996, 94:2228-34; and Makkar R R, Eigler N L, KaulS, Nakamura M, Forrester J S, Herbert J-M, Litvack F I, Effects ofclopidogrel, aspirin and combined therapy in a porcine ex vivo model ofhigh-shear induced stent thrombosis, Eur Heart J, 1998; 19:1538-1546.Stents were expanded on a tapered mandrel to an open diameter of 2.0 mmbefore being mounted in the perfusion chamber.

Extracorporeal Shunt and Perfusion Protocol

The extracorporeal shunt system utilized in this study has beenextensively characterized and described previously. See Kaul S, Makkar RR, Nakamura M, Litvack F, Shah P K, Forrester J S, Hutsell T, Eigler NL, Inhibition of Acute Stent Thrombosis under High-Shear Flow Conditionsby a Nitric Oxide Donor. DMHD/NO: An Ex-Vivo Porcine Arteriovenous ShuntStudy, Circulation, 1996, 94:2228-34; Makkar R R, Litvack F, Eigler N L,Nakamura M, Ivey P A, Forrester J S, Shah P K, Jordan R E, Kaul S,Effects of GP IIb/IIIa Receptor Monoclonal Antibody (7E3), Heparin, andAspirin in an Ex Vivo Canine Arteriovenous Shunt Model of StentThrombosis, Circulation, 1997; 95(4): 1015-1021; and Makkar R R, EiglerN L, Kaul S, Nakamura M, Forrester J S, Herbert J-M, Litvack F I,Effects of clopidogrel, aspirin and combined therapy in a porcine exvivo model of high-shear induced stent thrombosis, Eur Heart J, 1998;19:1538-1546. After a 60-minute stabilization period, stents weremounted in the tubular chamber and perfused with normal saline for 60seconds at 37° C. With a switch valve used to prevent stasis, blood wascirculated through the system, and flow was regulated at 100 mL/min for20 minutes by using a peristaltic pump (Masterfiex, Cole-PalmerInstrument Co.) placed in the circuit distal to the perfusion chamber.

The selected flow rate generates a wall shear rate of 2100 s⁻¹ at thestent surface. Shear rates were calculated according to the formula forlaminar flow of homogeneous Newtonian fluid in a cylindrical tube: shearrate=4.Q/π·R3, where Q is volume flow and R is radius. See Goldsmith HL, Turitto V T, Rheological aspects of thrombosis and hemostasis: basicprinciples and applications, Thromb Haemost, 1986;55:415-435. At highshear rates, as used in this study, blood is considered to beessentially a Newtonian fluid.

At the end of the perfusion period, saline was circulated through thechamber and ex vivo system for several minutes at 40 mL/min to clear anyvisible blood before another stent was mounted. At the completion ofeach perfusion period, the stents (weighed prior to perfusion) wereremoved from the chamber, dried, and weighed immediately. Thrombusweight was calculated as a difference between pre- and post-perfusionstent weights.

The top of each stent was covered with a heterologous porcine aorticstrip (Pel Freeze, Kans.) from which the intimal layer was removed tosimulate the thrombogenic conditions induced by vascular injuryassociated with stent implantation. The number of stent perfusion runsexamined varied from 6 to 8 during each experiment. Digital images ofstents were obtained with a Nikon 950 digital camera, downloaded into aPC and processed with image analysis software (PhotoShop Adobe 5.0). Asample of such images may be seen in FIG. 2( b).

At the end of each treatment, control stents were perfused to ensurereturn of stent thrombus weights towards baseline pre-treatment values.Effects on thrombus weight (TW), whole-blood platelet aggregation (PA),bleeding time (BT), activated clotting time (ACT), serum magnesiumlevel, and complete blood count (CBC) were quantified at various timepoints as shown in the protocol schematic. Mean arterial blood pressure(MABP) and heart rate (HR) were monitored and recorded throughout theprotocol.

Thirty minutes after administration of the heparin (in the heparinalone) or magnesium, 3 mL venous blood was collected in a siliconizedtest tube containing 0.3 mL of 0.129 molar sodium citrate or sodiumheparin (Becton Dickinson Vacutainer System). Whole blood aggregometry(Chronolog Corp.) was used to measure collagen (2 and 5 μg/mL)- andAdenosine diphosphate (2.5 μM)-induced platelet aggregation. Aggregationwas expressed as maximal increase in electrical impedance measured inohms at 6 minutes after the addition of agonist. The baseline plateletaggregation was 25±3 ohms. The platelet aggregation after the additionof Mg-late alone and heparin alone was 22±3 and 24±6 ohms, respectively.As noted above, data points in the Mg-treated animals were examinedwithin 20 minutes post-bolus (Mg-early) and >40 minutes post-bolus(Mg-late). The platelet aggregation obtained within 20 minutespost-bolus was 23±4 ohms. The platelet aggregation obtained >40 minutespost-bolus was 21.0±5 ohms.

Bleeding time is defined as the time from the creation of an incision tothe point where bleeding from the incision ceases. In the experimentaccording to one embodiment of the present invention discussed herein,bleeding time was measured from an incision on the ventral surface ofthe thigh with a No. 11 surgical knife.

In the experiment discussed herein that was conducted according to oneembodiment of the present invention, ACT was determined using aHemochron 400 (International Technidyne Corp.) machine in standardfashion. See Makkar R R, Litvack F, Eigler N L, Nakamura M, Ivey P A,Forrester J S, Shah P K, Jordan R E, Kaul S, Effects of GP IIb/IIIaReceptor Monoclonal Antibody (7E3), Heparin, and Aspirin in an Ex VivoCanine Arteriovenous Shunt Model of Stent Thrombosis, Circulation, 1997;95(4): 1015-1021; and Makkar R R, Eigler N L, Kaul S, Nakamura M,Forrester J S, Herbert J-M, Litvack F I, Effects of clopidogrel, aspirinand combined therapy in a porcine ex vivo model of high-shear inducedstent thrombosis, Eur Heart J, 1998; 19:1538-1546.

Serum magnesium levels for the test subjects were measuredspectrophotometrically using the magon dye method. See Elm R J,Determination of serum magnesium concentration by clinical laboratories,Magnes Trace Elem, 1991-92:60-6.

FIG. 1 illustrates a perfusion protocol that was run in an ex-vivoporcine shunt model according to an embodiment of the present invention.As used in FIG. 1, PA means platelet aggregation, ACT means activatedclotting time, BP means arterial blood pressure, and CBC means completeblood count. In order to obtain control thrombus weight, two to threestents were perfused in each animal prior to the administration of anydrug.

The stents were perfused pre- and post-treatment with heparin ormagnesium in a random fashion. Heparin was administered as 50 U/kg IVbolus followed by 25-50 U/kg/hr IV to keep ACT >150 seconds. Magnesiumsulfate (MgS04) was administered as a 2 gm bolus IV over 20 minutesfollowed by 2 gm/hour maintenance infusion. To assess a potentialtime-dependent antithrombotic effect of magnesium, data points wereexamined within 20 minutes post-bolus (Mg-early) and >40 minutespost-bolus (Mg-late). The time-dependent effects of magnesium were alsoexamined in a random fashion.

As shown in FIG. 1, the “pre-treatment” readings were taken from a stentthat was expanded to 2 mm in diameter in a tubular perfusion chamberinterposed in the shunt and perfused with blood at a shear rate of 2100s⁻1 for 20 minutes, without any magnesium or heparin in the system. The“heparin alone” readings were taken from stents that were perfused withblood for 20 minutes when the animals had been treated with intravenousheparin as described above. The perfusion study for the “heparin alone”reading was performed between twenty and thirty minutes after theadministration of heparin. Both the “heparin+Mg-Early” and“heparin+Mg-Late” readings were taken from stents that were perfusedwith blood for 20 minutes after the animals had been treated withintravenous heparin and magnesium as noted above. The perfusion periodfor the heparin+Mg-early readings started within 20 minutes afteradministration of the MgSO₄ bolus. The perfusion period for the heparin+Mg-late readings started more than 40 minutes after the administrationof the MgSO₄ bolus.

In one embodiment of the present invention, the magnesium bolus isfollowed by a 1 to 2 gm/hour maintenance infusion. For example, in theexperiment discussed in FIG. 1 herein, a 2 gm bolus IV was administeredto the test subject over 20 minutes followed by a 2 gm/hour maintenanceinfusion for an average magnesium dosage of 9 gm in a 30-kg pig (thedosages ranged from 5-13 gm).

The magnesium dosage required to achieve the benefits of one embodimentof the present invention is generally between 0.16 gm and 0.4 gm perkilogram body weight. A human weighing 100-kg would preferablyintravenously receive an approximately 2 gm bolus IV for roughly 10-20minutes. This would preferably be followed by an approximately 2 gm/hourmaintenance infusion for roughly 8-12 hours. It should be understood byone skilled in the art, however, that these dosages and times ofadministration may be varied depending on a number of factors including,but not limited to, the size of the subject. That is, a bolus dose of1-4 gm over 10-20 minutes may be administered, followed by a maintenancedose of 1-4 gm/hr. It should be noted that at higher bolus andmaintenance doses greater anti-thrombotic effects are observed. However,higher dosages also tend to affect heart rate and blood pressure.

FIG. 2( a) provides a bar graph showing the effect on thrombus weight indifferent treatment groups in an experiment according to one embodimentof the present invention. FIG. 2( b) provides side-on views of nitinolstents depicting thrombus burden under the various treatment conditions.Values are given as mean±SD. Eight to 15 stents were tested in theMagnesium alone groups. The number of stents varied between 20 and 35.

As may be seen from FIG. 2( a), the category of stents labeled“pre-treatment” has the greatest amount of stent thrombosis. Magnesiumalone (both early and late groups) as well as heparin reduce thrombusformation slightly. Heparin with magnesium-early reduces the thrombosismore significantly, and heparin with magnesium-late virtually preventedthrombus formation.

FIG. 3 provides a graphical depiction of the effects according to oneembodiment of the present invention of magnesium on heart rate and meanarterial blood pressure (MABP). In this experiment, magnesium in theform of magnesium sulfate was administered intravenously to the subjectanimals. Values are again provided as mean±SD. As may be seen from FIG.3, magnesium had no statistically significant effects on either heartrate or MABP.

The graph in FIG. 3 was generated by taking between 10 and 14observations at each time point. The MABP was measured in millimeters ofmercury (mmHg). The heart rate was measured in beats per minute (BPM).As may be seen from the graph, no statistically significant changes inheart rate and blood pressure were observed using ANOVA.

FIG. 4 provides a chart detailing the reduction in stent thrombosisbased on five differing stimuli according to one embodiment of thepresent invention. Particularly, FIG. 4 shows the effects of treatmentwith magnesium alone, heparin alone as well as combined treatment withmagnesium and heparin on acute stent thrombosis. The data shown in FIG.4 was obtained from an experiment similar to that which was discussed inFIGS. 2( a) and (b).

The total weight of a stent that was perfused with blood without theaddition of heparin or magnesium was 20±4 mg is referred to as stent TW.As shown in FIG. 1, the “pre-treatment,” or baseline stent TW, readingswere taken from a stent that was perfused with blood for 20 minutes,without any magnesium or heparin in the system. This number was used tocalculate the reduction in thrombus formation. The reduction in thrombusformation was calculated by subtracting the weight of a post-treatmentstent (post-heparin or magnesium treatment) from the average weight of astent that had been perfused with blood without the addition of heparinor magnesium.

As shown in FIG. 4, stent TW was reduced by 42±21%, 47±19%, 48±16% inthe Mg-early, Mg-late and heparin-treated group, respectively. Theweight was reduced by 67±12% in the heparin+Mg-early-treated group. Thethrombus weight was further reduced by 86±8% in theheparin+Mg-late-treated group. All five of these calculations had aP<0.001 versus pretreatment. (P<0.05 heparin+Mg-late versus heparin andMg-early; P<0.01 heparin+Mg-early and Mg-late versus heparin alone,Mg-early and late alone, ANOVA). Magnesium had no significant effects onplatelet aggregation, activated clotting time and bleeding time. Theserum Mg level was the only variable that correlated with TW (r=−0.70.P.002). There were no significant effects on heart rate or mean arterialblood pressure.

As may be seen in FIG. 4, the antithrombotic effects of combinedtreatment with heparin and Mg were significantly more pronouncedcompared with magnesium or heparin alone (P<.001, ANOVA).Heparin+Mg-late produced a slightly greater, and statisticallysignificant, reduction in TW compared with heparin+Mg-early group(P<0.05, ANOVA).

FIG. 5 provides a chart detailing the effects of the heparin andmagnesium as used in an experiment according to one embodiment of thepresent invention on 5 μg/mL collagen-induced platelet aggregation,bleeding time and ACTs.

As shown in FIG. 5, the reduction in whole-blood platelet aggregationwith the addition of magnesium, from 25±3 to 21±5 ohms, was notsignificant. To exclude the possibility that minimal magnesium effect onplatelet aggregation may be related to the citrate anticoagulant usedwhich may influence ionic concentrations because of calcium chelation inthe samples, aggregation was tested in heparinized as well as citratedblood in 3 pigs. Magnesium produced no significant inhibitory effect onplatelet aggregation in heparin-stabilized samples (26±2 pre- and 27±2post-Mg) as compared to citrate-stabilized blood (25±3 pre- and 22±3post-Mg). Platelet aggregation was, however, slightly enhanced inheparinized samples. There was no effect of magnesium on plateletaggregation in response to either a lower concentration of collagen-2μg/ml or to another platelet agonist—ADP (2.5 μM) (data not shown).Therefore, magnesium had no significant effect on platelet aggregation,regardless of the anticoagulant used to stabilize blood.

As shown in FIG. 5, heparin increased the bleeding time from 4.0±0.5 to5.3±0.6. Heparin also prolonged ACT from 109±8 seconds to 185±36 seconds(P<0.01. ANOVA). Magnesium had no significant effects on either bleedingtime or ACT beyond the heparin effect. There were no episodes ofsignificant bleeding in any of the animals studied. Treatment withheparin or magnesium had no significant effects on either platelet orwhite blood cell counts or hematocrit (data not shown).

As further shown in FIG. 5, serum magnesium levels were higher in themagnesium-treated animals compared to control. However, the levels werevirtually similar in the Mg-early and Mg-late group. Serum magnesiumcorrelates significantly with thrombus weight (r=−0.70. P=0.002),bleeding time (r=0.54, P=0.05) and heart rate (r=−0.55, P=0.002). Serummagnesium was the only variable that correlated significantly withthrombus weigh. No significant correlation of thrombus weight wasobserved with platelet aggregation (r=0.4, P=0.07).

As previously noted, the timing of the administration of magnesiumweighs significantly on the benefit seen in reduced thrombosis weight.Heparin+Mg-early produced a significant reduction in thrombosis weightwhen compared to the thrombus weight for heparin alone or thepre-treatment group. However, heparin+Mg-late produced an even greaterreduction in thrombosis weight. The difference in antithrombotic effectin Mg-early and Mg-late group cannot be solely explained by adose-dependent effect since serum magnesium levels were notsignificantly different. However, serum magnesium does not accuratelyreflect intracellular levels of magnesium. Given the increased exposureto magnesium, it is likely that the intracellular levels of Mg may behigher in the Mg-late as compared with Mg-early tests despite similarserum Mg levels.

Human Testing

One embodiment of the present invention was tested in human subjects,with very positive results. Based on these tests, the use of magnesiumwas determined to be beneficial to prevent in-stent thrombosis. Thiseffect was evident without any hemostatic or hemodynamic complications.

Twenty one low-risk patients undergoing non-acute percutaneous coronaryintervention with stent implantation received a two gm bolus ofintravenous MgSO₄ followed by a 1.5 gm/hour infusion for four hours anda 1 gm/hour infusion for the next eight hours. This test resulted in atotal administration of 16 gm of magnesium. The preset primary endpointsfor this experiment were: acute thrombotic occlusion and need forplatelet IIb/IIIa inhibitors bailout, death, myocardial infarction,recurrent ischemia and need for urgent revascularization at 48 hours and30 days. The secondary safety endpoints included hypotension,bradycardia, bleeding complications and heart block within the first 24hours.

In all cases, the interventions were finished with normal coronaryartery blood flow (TIMI 3 flow). There was no case of GP IIb/IIIainhibitors bailout. Death, myocardial infarction, and urgentrevascularization were also not seen. Serum magnesium levels increasedsignificantly from a 2.1±0.3 baseline to 3.2±0.3 mg/dL post-bolus(P<0.0001). Further, magnesium did not have a significant effect oneither heart rate or mean arterial blood pressure. There was no furthersignificant increase in magnesium concentration after completion of themagnesium sulfate infusion (the magnesium level post-infusion was3.5±0.8 mg/dL).

All of the data presented herein is presented as mean±SD. Thestatistical difference between means was determined by one-way ANOVA. Ifmeans were shown to be significantly different, multiple comparisons bypairs were performed by the Bonferroni test (Graphpad Prism version3.0). Spearman's correlation analysis was performed to explore therelationship between serum magnesium and other variables and betweenthrombus size and other variables. A value of P<0.05 was consideredsignificant.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention. The presently disclosedembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than the foregoing description,and all changes that come within the meaning and range of equivalency ofthe claims are therefore intended to be embraced therein.

1. A method of reducing stent thrombosis under high shear blood flowconditions in a subject, the method comprising: providing a stent coatedwith an effective amount of a magnesium-based compound to administer themagnesium-based compound; and inserting the stent within a bodypassageway in the subject.
 2. The method of claim 1, wherein themagnesium-based compound is selected from the group consisting ofmagnesium sulfate, magnesium oxide, magnesium phosphate, and magnesiumchloride.
 3. The method of claim 1, further including the step ofassociating the stent with an instrument for inserting the stent withina body passageway.
 4. The method of claim 3, wherein the instrument iscoated with a magnesium-containing compound to administer themagnesium-based compound.
 5. The method of claim 1, further includingthe step of administering a magnesium-based compound to a subject sothat a serum magnesium level between 3 and 5 mEq/L is attained prior toexposure of the subject to a stent-induced thrombogenic stimuli, whereinthe administering step is carried out prior to inserting the stentwithin the body passageway.
 6. The method of reducing stent thrombosisaccording to claim 5, wherein the serum magnesium level is attained atleast 15-20 minutes before the subject is exposed to the stent-inducedthrombogenic stimuli.
 7. The method of claim 1, further comprisingadministering an effective amount of a magnesium-based compound to thesubject between approximately 15 and 90 minutes prior to insertion ofthe stent.
 8. The method of claim 7, wherein the magnesium-basedcompound coated on the stent and the further administeredmagnesium-based compound are selected from the group consisting ofmagnesium sulfate, magnesium oxide, magnesium phosphate, and magnesiumchloride.
 9. The method of claim 7, wherein the magnesium-based compoundis administered intravenously.
 10. The method of claim 7, wherein themagnesium-based compound is administered as an injection.
 11. The methodof claim 7, wherein the magnesium-based compound is administered to thesubject between 30 and 60 minutes prior to insertion of the stent. 12.The method of claim 7, further comprising administering an effectiveamount of heparin to the subject.
 13. The method of claim 7, whereinadministering an effective amount of a magnesium-based compoundcomprises administering the magnesium-based compound as a 2 gm bolus.14. The method of claim 13, wherein the bolus is administered over a 10to 20 minute time period.
 15. The method of claim 13, wherein afteradministering the magnesium-based compound as a 2 gm bolus, the methodfurther comprises administering a 1 to 1.5 gm/hour maintenance infusionof a maintenance magnesium-based compound.
 16. The method of claim 15,wherein the maintenance infusion is administered after inserting thestent into the subject.
 17. The method of claim 15, wherein themaintenance infusion is administered over an 8 to 12 hour time period.18. The method of claim 15, wherein the maintenance magnesium-basedcompound is selected from the group consisting of magnesium sulfate,magnesium oxide, magnesium phosphate, and magnesium chloride.
 19. Themethod of reducing stent thrombosis according to claim 6, wherein thefurther administered magnesium-based compound is administeredintravenously.
 20. A method of reducing stent thrombosis under highshear blood flow conditions in a subject, the method comprising:administering a magnesium-based compound intravenously to a subject sothat a serum magnesium level between 3 and 5 mEq/L is attained prior toexposure of the subject to a stent-induced thrombogenic stimuli, whereinthe administering step is carried out prior to inserting the stentwithin the body passageway; providing a stent coated with an effectiveamount of a magnesium-based compound selected from the group consistingof magnesium sulfate, magnesium oxide, magnesium phosphate, andmagnesium chloride; associating the stent with an instrument coated witha magnesium-containing compound for inserting the stent within a bodypassageway; and inserting the stent within a body passageway in thesubject.
 21. The method of reducing stent thrombosis according to claim20, wherein the serum magnesium level is attained at least 15-20 minutesbefore the subject is exposed to the stent-induced thrombogenic stimuli.22. A method of reducing stent thrombosis under high shear blood flowconditions in a subject, the method comprising: providing a stent coatedwith an effective amount of a magnesium-based compound; inserting thestent within a body passageway in the subject; and administering aneffective amount of a magnesium-based compound to the subject betweenapproximately 15 and 90 minutes prior to insertion of the stent.
 23. Themethod of claim 22, wherein the magnesium-based compound coated on thestent and the magnesium-based compound administered prior to insertionof the stent are selected from the group consisting of magnesiumsulfate, magnesium oxide, magnesium phosphate, and magnesium chloride.24. The method of claim 22, wherein the magnesium-based compoundadministered prior to insertion of the stent is administeredintravenously.
 25. The method of claim 22, wherein the magnesium-basedcompound administered prior to insertion of the stent is administered asan injection.
 26. The method of claim 22, wherein the magnesium-basedcompound administered prior to insertion of the stent is administered tothe subject between 30 and 60 minutes prior to insertion of the stent.27. The method of claim 22, further comprising administering aneffective amount of heparin to the subject.
 28. The method of claim 22,wherein administering an effective amount of a magnesium-based compoundcomprises administering the magnesium-based compound as a 2 gm bolus.29. The method of claim 28, wherein the bolus is administered over a 10to 20 minute time period.
 30. The method of claim 28, wherein afteradministering the magnesium-based compound as a 2 gm bolus, the methodfurther comprises administering a 1 to 1.5 gm/hour maintenance infusionof a maintenance magnesium-based compound.
 31. The method of claim 30,wherein the maintenance infusion is administered after inserting thestent into the subject.
 32. The method of claim 30, wherein themaintenance infusion is administered over an 8 to 12 hour time period.33. The method of claim 30, wherein the maintenance magnesium-basedcompound is selected from the group consisting of magnesium sulfate,magnesium oxide, magnesium phosphate, and magnesium chloride.